This invention is in the field of sulfotransferase enzymes, and in particular assay methods for identifying agents that modulate sulfotransferase activity.
Sulfation of biomolecules is now appreciated as a major regulatory modification that affects interactions in the extracellular space. The sulfotransferases (STs) are a family of enzymes that catalyzes the sulfation of biomolecules. Many STs act on glycoproteins in the Golgi compartment. Dozens of STs have been discovered, and in some cases the epitopes they generate have been linked to disease processes. For example, specific carbohydrate sulfates govern growth factor binding and activation, and leukocyte adhesion during inflammation. Tyrosine sulfation is a prerequisite for HIV-1 binding to host cell co-receptors in addition to other cell adhesion events. These examples have piqued the interests of both biologists and medicinal chemists, as STs are emerging as attractive therapeutic targets.
There has been some progress in the development of relatively efficient assays for individual carbohydrate STs. A traditional architecture involves radiolabel transfer from 35S-labeled 3xe2x80x2-phosphoadenosine 5xe2x80x2-phosphosulfate (PAPS) to a carbohydrate substrate bearing a hydrophobic tail for capture on reversed-phase cartridges. While this approach can be adapted to 96-well format, it requires several labor-intensive washing and elution steps in order to purify the sulfated product before quantitation by liquid scintillation. Biotinylated substrates have been used for sulfotransferase assays, wherein capture with immobilized avidin separates the 35S-labeled products from unreacted 35S-labeled PAPS. Fewer washing steps are required, but microtiter plate-based systems that immobilize avidin are fairly expensive, particularly for high-throughput screens. Wong and co-workers reported an enzyme-coupled continuous spectrophotometric assay that could in principle be applied to any sulfotransferase. While ideal for kinetic assays, the presence of a second enzyme, in this case an aryl sulfotransferase, might complicate ST-targeted inhibitor screens.
Therefore, there is a general need for simple, high-throughput screening strategies for measuring activities in the presence and absence of inhibitors. The present invention addresses this need.
Literature
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The instant invention provides methods for identifying agents that modulate an enzymatic activity of a carbohydrate sulfotransferase. The methods generally involve contacting the sulfotransferase, in a reaction solution, with a sulfate donor, a test agent, and a polymeric sulfate acceptor that is readily separated from the reaction solution. Determination of an effect of the test agent on the sulfotransferase is by detecting the amount of sulfate in the polymeric sulfate acceptor that has been separated from the reaction solution. The invention further provides kits for use in carrying out the subject methods.